1. Field of the Invention
Single stranded DNA plays a vital role in a number of different technologies. For instance, the determination of nucleic acid sequence has had an enormous impact in the fields of molecular biology, biochemistry, and genetics. Many sequencing protocols require utilization or generation of single stranded DNA at some stage of the process. The need for sequencing reagents and automated sequencing instruments has also created significant commercial activity. Single stranded nucleic acids are also used as probes in the emerging medical diagnostics technology based on hybridization of nucleic acids to detect pathogens and disease or disease-susceptible states. In addition, DNA "fingerprinting" techniques utilizing single stranded DNA probes has improved forensic methodologies.
2. Description of Related Disclosures
The present invention provides a method for generating single stranded DNA by the polymerase chain reaction (PCR). The PCR procedure involves repeated cycles of denaturation of the DNA, annealing of oligonucleotides primers to sequences flanking the segment of interest, and primer extension, typically mediated by a DNA polymerase, resulting in a doubling of the amount of a specific DNA fragment with each cycle. The PCR process requires a primer pair; the two primers define a segment of double stranded DNA that accumulates as the PCR product. The PCR process is described in U.S. Pat. No. 4,683,202 and results in a myriad number of identical copies of a double stranded DNA fragment.
PCR can be used to clone nucleic acid sequences never before identified or sequenced. The sequence of the PCR product can be identified either indirectly by hybridization to oligonucleotide probes, as described in U.S. Pat. No. 4,683,195, or directly by determination of the nucleotide sequence of the PCR amplified target. The sequence of DNA fragments generated by PCR has previously been determined either by cloning the fragments into derivatives of bacteriophage M13, as described by Scharf et al., 1986, Science 233:1076-1078, and Horn et al., 1988, Proc. Natl. Acad. Sci. USA 85:6012-6016 or by direct sequencing of the double stranded template, using a third "internal" primer, as described by Wong et al., 1987, Nature 330:384-386, and Wrischnik et al., 1987, Nuc. Acids Res. 15:529-535. However, the M13 cloning method is time consuming and requires that several sequences be determined to distinguish mutations occurring in the original sequence from i) random point mutations introduced by lack of fidelity of the DNA polymerase; and ii) artifacts such as the formation of mosaic alleles by in vitro recombination. Direct sequencing of double stranded templates can present difficulties due to the rapid reannealing of strands and the presence of sequences partially homologous to that of the sequencing primer on both strands, resulting in compound sequence ladders. These problems can be overcome by the method of the present invention to modify the PCR reaction in such a way that an excess of full length single stranded DNA of a chosen strand is produced that is suitable for sequence determination.